Localization of Specific Epitopes on Human Microtubule-Associated Protein 2

Abstract: Microtubule-associated protein 2 (MAP-2) is an abundant neuronal cytoskeletal protein that binds to tubulin and stabilizes microtubules. Using fusion protein constructs we have defined the epitopes of 10 monoclonal antibodies (mAbs) to discrete regions of human MAP-2. Proteins were expressed in pATH vectors. After electrophoresis, immunoblotting was performed. By western blot analysis five of the mAbs (AP-14, AP-20, AP-21, AP-23, and AP-25) share epitopes with only the high molecular weight isoforms (MAP-2a, MAP-2b); two of the mAbs (AP-18 and tau 46) recognize MAP-2a, MAP-2b, and MAP-2c. Although AP-18 immunoreactivity was detected within heat-stable protein homogenates isolated from a human neuroblastoma cell line MSN, fusion protein constructs encompassing human MAP-2 were negative, suggesting that the AP-18 epitope is phosphorylated. Furthermore, AP-18 immunoreactivity was lost after alkaline phosphatase treatment of heat-stable protein preparations from MSN cells. Four of the mAbs (322, 636, 635, and 39) recognize epitopes located within amino acids 169–219 of human MAP-2. AP-21 maps to a region between amino acids 553 and 645. AP-23 maps between amino acids 645 and 993, whereas AP-20, AP-14, and AP-25 map between amino acids 995 and 1332. Expression of the region of MAP-2 between amino acids 1787 and 1824 was positive to tau 46.     

Immunocytochemical Localization of the Vacuolar H-ATPase in Maize Root Tip Cells

The vacuolar H⁺-ATPase of maize (Zea mays L.) root tip cells has been localized at the EM level using rabbit polyclonal antibodies to the 69 kilodalton subunit and protein A-colloidal gold. Intracellular gold particles were detected mainly on the tonoplast and Golgi membranes. Only about 27% of the vacuoles were labeled above background. The absence of gold particles on the majority of vacuoles suggests either that the tonoplast H⁺-ATPase is degraded during tissue preparation or that the small vacuoles of root tip cells are specialized with respect to H⁺-ATP ase activity. The pattern of gold particles on the labeled vacuoles ranged from uniform to patchy. Virtually all of the Golgi bodies were labeled by the antibody, but the particle densities were too low to determine whether the H⁺-ATPase was associated with specific regions, such as the trans-face. Cell wall-labeling was also observed which could be partially prevented by the inclusion of gelatin as a blocking agent. The immunocytochemical results confirm previous biochemical studies with isolated membrane fractions (A Chanson, L Taiz 1985 Plant Physiol 78: 232-240).     

Mucilage on the Root Surface and Root Hairs of Sorghum: Heterogeneity in Structure, Manner of Production and Site of Accumulation

Small drops of a mucilaginous character near the tip of roothairs were seen by light microscopy in several species of Sorghumand the Sorghum hybrid Vidan. Electron microscopy revealed thatthe drops are formed from at least two distinct substances,both apparently secreted from the endoplasmic reticulum. In addition, a patchy, fibrillar mucilaginous layer, also withat least two components, was found on the cell wall of the roothairs and on the outer wall of ordinary root epidermal cells.Golgi bodies as well as mitochondria take part in its production.As a rule, the mucilaginous patches are colonized by bacteria. Sorghum, root hairs, mucilage     

Localization of Tumour Specific Antigen on External Surface of Plasma Membrane

FOLLOWING the adventitious finding that lymphocytes from patients with malignant (but not benign) neoplasia are sensitized to encephalitogenic basic protein of myelin (EF)¹ and to a protein similarly extracted from a variety of tumours², a logical step would be to determine the subcellular localization of the specific antigen(s) on or within the cancer cell and whether or not the antigen continues to be associated with human malignant (or transformed) cells long maintained in culture. If this were so, then such cultures might serve as a convenient and standard source for an antigen of considerable potential in the diagnosis—and perhaps also the treatment—of malignant disease. The allied question of the identity and uniqueness of the antigens extractable from different cell lines or tumours will be the subject of a separate communication.     

Colonization of Wheat Root Hairs and Roots by Agrobacteria

Formation of extracellular structures in pure culture and in interaction with wheat root surface was studied by scanning and transmission electron microscopy. The effects of various factors (growth temperature as well as pretreatment of agrobacteria with kalanchoe extract, acetosyringone, and centrifugation) on formation of extracellular structures was tested. The data on Agrobacterium tumefaciens (wild-type strain C58 and mutants LBA2525 (virB2::lacZ) and LBA288 (without the Ti plasmid)) adhesion to wheat root surface and root hairs after pretreatment of agrobacteria with inducer of virulence genes (vir) acetosyringone were obtained. Formation of agrobacterial cell aggregates on wheat root hair tips was demonstrated. The proportion of root hairs with agrobacterial aggregates on the root hair tip insignificantly changed after pretreatment with acetosyringone but considerably increased after treatment of A. tumefaciens C58 and LBA2525 with kalanchoe leaf extract. The most active colonization of root hairs and formation of agrobacterial aggregates on hair root tips was observed at 22°C. The capacity of agrobacteria for adhesion on monocotyledon surface could be changed by pretreatment of bacteria with various surface-active substances. Bacterial cells subjected to centrifugation had a decreased capacity for attachment to both wheat root surface and root hairs. The relationship between the capacity for adhesion and pilus production in agrobacteria was considered.     

Cytokinin Inhibits Lateral Root Development at the Earliest Stages of Lateral Root Primordium Initiation in Maize Primary Root

Root architecture is basically controlled by auxin and cytokinin, which antagonize in the formation of lateral roots (LRs) along the primary root (PR) axis. Several mechanisms have been proposed to explain the interaction between these two hormones, cytokinin being the hormone that inhibits LR formation. The analysis of the cytokinin effect on LR formation using LRs in several stages of development could indicate which steps of LR formation are more sensitive to cytokinin. The application of cytokinin to maize PRs showed that the inhibitory effect of cytokinin on LR formation was greater in the zones in which the initial events to form new LRs are taking place. In the presence of cytokinin, the PR is not able to produce new LRs in the initiation zone; this inhibitory effect is permanent as this zone did not recover the capability to form LRs after removing cytokinin. However, the LR density in zones with appreciable LR primordia when cytokinin was applied was only slightly inhibited when a high concentration was used. These results showed that LR formation is more sensitive to the inhibitory effect of cytokinin in the earliest stages of LR development. However, the elongation of a LR primordium to emerge and the subsequent elongation of the new LR were only slightly affected by cytokinin.

     

Studies on the Secretion of Maize Root Cap Slime: II. Localization of Slime Production

The distribution of fucose-containing polysaccharides in apical 1-cm sections of corn (Zea mays cv. SX-17) root tips was analyzed. Fucose-containing polysaccharides were localized predominantly in the apical 1 mm of the root, i.e., in the apical initials and root cap. An analysis of the distribution of incorporated radioactive label from l-fucose[(3)H] gave similar results. After a 2-hr incubation with fucose[(3)H], label was found principally in two components, namely a water-soluble slime fraction and hemicellulose. The incorporation of fucose into the water-soluble, ethanol-insoluble fraction was primarily in the apical 1 mm of the root, whereas incorporation into a water-insoluble, potassium hydroxide-soluble fraction was in the region 2 to 5 mm behind the root cap. Addition of sucrose to the incubation medium during fucose[(3)H] incorporation reduces label uptake but increases the amount of label in the fucose-rich secreted polysaccharide. The utility of fucose as a marker for the secreted polysaccharide was confirmed by demonstrating that no appreciable metabolism of this sugar occurs.     

The Infection of Trifolium subterraneum Root Hairs by Rhizobium trifolii

Both host cultivar and Rhizobium strain influence the numberof infected root hairs of Trifolium subl-errctneum, seedlings;Yarloop had fewer infections than Cranmore, Mount Barker, orTallarook and Rhizobium trifolii strain 5 infected fewer hairsthan strain TA1. Hybrid lines bred for sparse or abundant nodulationhad similar numbers of infected hairs, but. as in the cultivars,these always greatly exceeded the number of nodules formed.More infection threads aborted early during growth in the roothairs of Cranmore than in other hosts and early abortion wasmore common with strain 5 than strain TA1 In all hosts and with both Rhizobium strains, infection beganon day 3 and was initially restricted to one or two zones alongthe root with later infections extending these zones or initiatingnew ones. The exponential rate of infection (least for Yarloop)slows sharply when nodules appear. Early nodules and lateral roots formed at different places indifferent hosts, and in most cultivars and hybrid lines nodulesand laterals occurred in mutually exclusive zones. Primordiaarising above the first nodule failed to develop.     

Abscisic Acid Elicits the Water-Stress Response in Root Hairs of Arabidopsis thaliana

Water stress has been shown to cause root hairs to become short and bulbous. Because abscisic acid (ABA) mediates a variety of water-stress responses, we investigated the response of Arabidopsis thaliana root hairs to ABA. When wild-type root hairs were treated with ABA, they exhibited the water-stress response. The Arabidopsis mutants abi1 and abi2, which are insensitive to ABA at the seedling stage, did not display the root hair response. These data suggest that ABA may mediate the response of root hairs to water stress. The drought response of root hairs resulting in an inhibition of tip growth will provide an easy screen to select mutations that are insensitive to ABA and/or involved in tip growth.     

Effect of Chilling on DNA Endoreplication in Root Cortex Cells and Root Hairs of Soybean Seedlings

Relative nuclear DNA contents in cortex parenchyma cells in root segments of 3- and 7-d-old soybean seedlings grown at 25 °C and in plants grown for 3 d at 25 °C, and then for 4 d at 10 °C, were determined with cytophotometry. Measurements revealed that in each variant the cortex cell nuclei with DNA content between 2C and 8C were in all the examined segments and nuclei with 8C – 16C DNA appeared in higher parts of roots. However, in chilled plant cells the number of 8C – 16C DNA nuclei was very low. Therefore, chilling inhibited endoreplication in comparison with plants grown at 25 °C for 7 d, and even reduced endopolyploidy level as compared to the initial seedlings, i.e. 3-d-old plants. DNA contents in root hairs grown at 25 °C (control) and in root hairs emerged at 10 °C were also determined. In controls 4C – 8C DNA nuclei predominated while in chilled plants an additional population of 2C – 4C DNA appeared. Thus a reduction of DNA synthesis was brought about by low temperature. The occurrence of an intermediate DNA contents besides those with full endoreplication cycles suggests the possibility of differential DNA replication. This suggestion seems to be supported by the lack of ³H-thymidine incorporation into root hair nuclei at the examined developmental stage both in control and chilled root hairs. The same number, but larger, chromocentric lumps in polyploid cortex cell nuclei of higher root zones, in comparison to meristematic nuclei, suggests that endoreduplication process occurred. This revised version was published online in July 2006 with corrections to the Cover Date.     

Absence of Root Hairs in Non-Nodulating Groundnut, Arachis hypogaea L.

Root hairs observed at the site of lateral root emergence innodulating groundnut cultivars were found to be absent in non-nodulatinggroundnut lines. In a segregating F₂-population of the crossNC 17 x PI 259747 a strong association was observed betweenthe presence of root hairs and nodulation, and the absence ofroot hairs and non-nodulation. Key words: Root hairs, Arachis hypogaea, Non-nodulation     

Leaf Pubescence: The Significance of Lower Surface Hairs for the Spectral Properties of the Upper Surface

The fraction of the global radiation incident on the upper surfaceof leaves of Tussilago farfara L. which is reflected, variesaccording to the presence or absence of hairs on the lower surface.Calculations of the radiation fluxes at 550 and 1000 nm wavelengthsprove that these differences arise from a partial reflectionof the radiation emerging from the lower epidermis by the haircover.     

A Study of 'Exudation' from the Root Hairs of Apple Roots by Time-lapse Cine-photomicrography

The growth of root hairs and the occurrence of droplets of liquidon the surface of a young apple root growing behind a plateglass window, were investigated by making a time-lapse film.Spherical droplets of liquid enlarged on the tips of individualroot hairs, and later coalesced to form large globules. Afterfour days some of the larger globules shrank and were then seento be covered with a transparent skin.     

Role of Root Hairs and Lateral Roots in Silicon Uptake by Rice

The rice plant (Oryza sativa L. cv Oochikara) is known to be a Si accumulator, but the mechanism responsible for the high uptake of Si by the roots is not well understood. We investigated the role of root hairs and lateral roots in the Si uptake using two mutants of rice, one defective in the formation of root hairs (RH2) and another in that of lateral roots (RM109). Uptake experiments with nutrient solution during both a short term (up to 12 h) and relatively long term (26 d) showed that there was no significant difference in Si uptake between RH2 and the wild type (WT), whereas the Si uptake of RM109 was much less than that of WT. The number of silica bodies formed on the third leaf in RH2 was similar to that in WT, but the number of silica bodies in RM109 was only 40% of that in WT, when grown in soil amended with Si under flooded conditions. There was also no difference in the shoot Si concentration between WT and RH2 when grown in soil under upland conditions. Using a multi-compartment transport box, the Si uptake at the root tip (0-1 cm, without lateral roots and root hairs) was found to be similar in WT, RH2, and RM109. However, the Si uptake in the mature zone (1-4 cm from root tip) was significantly lower in RM109 than in WT, whereas no difference was found in Si uptake between WT and RH2. All these results clearly indicate that lateral roots contribute to the Si uptake in rice plant, whereas root hairs do not. Analysis of F(2) populations between RM109 and WT showed that Si uptake was correlated with the presence of lateral roots and that the gene controlling formation of lateral roots and Si uptake is a dominant gene.